Código C para microprocesador MicroBlaze

(Heptadecafluoro-1,1,2,2-tetrahydrodecyl)trimethoxysilane (17FTMS) was purchased from Gelest (Morrisville, PA). Methyltrimethoxysilane (MTMOS) was purchased from Fluka (Buchs, Switzerland). Griess assay reagents were purchased from Promega (Madison, WI). Tryptic soy broth (TSB) and brain heart infusion (BHI) broth were purchased from BD Biosciences (San Jose, CA). Dulbecco’s Modified Eagle Medium (DMEM), McCoy’s Medium 5A Modified, fetal bovine serum (FBS), dipotassium ethylenediaminetetraacetic acid (K2EDTA), nicotinamide adenine dinucleotide phosphate (NADPH), and nitrate reductase (from Aspergillus niger) were purchased from Sigma (St. Louis, MO). Opti-MEM I (a reduced serum medium) was purchased from Life Technologies (Grand Isle, NY). Leibovitz medium (L-15; a carbon dioxide-free cell culture medium) was purchased from Lonza (Basel, Switzerland). Porcine blood was obtained from the Francis Owen Blood

Research Laboratory (University of North Carolina; Chapel Hill, NC). Blood serum was obtained by collecting porcine blood without the addition of anticoagulant. After allowing the blood to clot, it was centrifuged at 2500 rpm for 15 min and the supernatant (i.e., serum) was removed. To obtain blood plasma, porcine blood was drawn into a tube with K2EDTA (~1.8 mg mL-1), mixed immediately, and then centrifuged at 2500 rpm for 15 min. After centrifugation, three layers were present (from top to bottom: plasma, leukocytes, and erythrocytes); the top layer was removed. A Millipore Milli-Q UV Gradient A10 System (Bedford, MA) was used to purify distilled water to a final resistivity of 18.2 MΩ·cm and a total organic content of ≤6 ppb. Nitrogen and argon gases were purchased from AirGas National Welders (Raleigh, NC). Nitric oxide gas was purchased from Praxair (Danbury, CT). Other solvents and chemicals were analytical-reagent grade and used as received. 2.2.1 Preparation of physiological media

Artificial saliva solution was prepared as described by Arvidson et al.34 via the addition of the following components to 500 mL of Milli-Q water: 279 mg monopotassium phosphate, 284 mg sodium phosphate dibasic, 1.24 g potassium bicarbonate, 482 mg sodium chloride, 254 mg magnesium chloride, 184 mg calcium chloride, and 394 mg citric acid. The pH of the solution was subsequently adjusted to 6.7. A 500 mL solution of artificial normal human urine was prepared according to Kark et al.35 by adding the following to Milli-Q water: 2.40 g sodium phosphate, 2.25 g potassium chloride, 3.75 mg sodium chloride, and 9.1 g urea. After the pH of this solution was adjusted to 5.9, 25 mg bovine serum albumin and 1.0 g creatinine were added. For a 500 mL solution of physiosol in Milli-Q water the following were added: 2.63 g sodium chloride, 185 mg potassium chloride, 150 mg magnesium chloride, 1.11 g sodium gluconate, and 2.51 g sodium acetate.36 The pH of the

solution was then adjusted to 6.0. A saturated NO solution (1.9 mM NO) was made by purging ~20 mL of PBS with argon for 30 min to remove oxygen, followed by NO gas for 20 min.

2.2.2 Synthesis of PROLI/NO

N-diazeniumdiolated L-proline (PROLI/NO) was prepared following a previously published protocol.37 Briefly, L-proline (2.05 g) was dissolved in a solution of methanol (25 mL) and sodium methoxide (2.00 g). The solution was then placed in a stainless steel reaction vessel and flushed with Ar six times (three in succession, three for 10 min each), then charged with NO at a pressure of 10 atm for 3 d with constant stirring. Six additional Ar purges were performed after 3 d. The solution was then precipitated by the addition of diethyl ether (150 mL) at −20 °C for 4 h. The white precipitate was isolated by vacuum filtration and dried in vacuo to yield PROLI/NO, which was stored at −20 °C until use. Ultraviolet spectra of a 14.9 μg·mL–1

solution of the product (in 1.0 M sodium hydroxide) were acquired on a Thermo Scientific evolution array UV–visible spectrophotometer (Figure 2.1). The molecular weight of pure PROLI/NO was taken to be 251 g·mol–1

. 2.2.3 Griess assay

To quantify NO via the Griess assay,38 50 µL of a 2 mg mL-1 solution of PROLI/NO in 100 mM sodium hydroxide (NaOH) was added to 15 mL of desired media and incubated at room temperature for at least 24 h. Aliquots (50 µL) of this sample were added to a sulfanilamide solution (50 µL) and incubated in the dark at room temperature for 5 min. Naphthylethylenediamine (50 µL) was added to the mixture to form a colorimetric product with concomitant absorbance measured in each well at 540 nm using a LabSystems

MultiSkan RC microplate reader (Helsinki, Finland). Sodium nitrite standards were used to normalize the assay reactivity and associated absorbance.

For analysis of blood constituents (i.e., plasma and serum), NADPH (25 µL) and nitrate reductase (2 µL) were added to the samples and allowed to incubate for at least 30 min prior to the addition of the Griess reagents.

2.2.4 Chemiluminescence detection

Real-time NO release was monitored using a Sievers 280 Chemiluminescent NO Analyzer (Boulder, CO). The instrument was calibrated with a 25.6 ppm gas standard (balance N2) and an atmospheric sample that had been passed through a NO zero filter. Samples were prepared by adding 10 µL of a 2 mg mL-1 solution of PROLI/NO in 100 mM NaOH to 30 mL of desired media that had been degassed in a sample vessel for at least 20 min. Nitric oxide produced in the vessel was carried to the NO analyzer by a stream of nitrogen gas bubbled into the solution (80 mL min-1) across the headspace of the flask (120 mL min-1), equivalent to 200 mL min-1 flow to the instrument.

2.2.5 Electrochemical detection

Inlaid 2 mm diameter polycrystalline platinum (Pt) disk electrodes sealed in Kel-F (CH Instruments; Austin, TX) were mechanically polished with successively finer grades of deagglomerated alumina slurries down to 0.05 µm particles (Buehler; Lake Bluff, IL). Residual alumina was removed using an ultrasonic cleaner (in water) and the electrodes were dried with nitrogen. A fluorinated NO selective xerogel membrane was applied to the electrode as previously described to minimize response to common interferents.39,40 Briefly, a silane solution was prepared by mixing MTMOS (60 µL) in ethanol (300 µL). To this solution, 17FTMS (15 µL) was added, resulting in a 20% v/v fluoroalkoxysilane (balance

MTMOS) mixture. The silane solution was subsequently mixed with water (80 µL) and 0.5 M HCl (5 µL) for 1 h. The resulting sol (1.5 µL) was cast onto Pt working electrodes and allowed to cure for 24 h under ambient conditions. To evaluate the analytical performance of the NO sensors, amperometric measurements were performed using a CH Instruments 730B bipotentiostat (Austin, TX). The electrode assembly (3-electrode configuration) consisted of the xerogel-modified Pt working electrode, a Pt-coiled counter electrode, and a Ag/AgCl reference electrode (3.0 M KCl; CH Instruments). Electrooxidation currents were recorded at an applied potential of +700 mV (vs. Ag/AgCl). To measure NO release in the various media using PROLI/NO as the NO source, a 2 mg mL-1 solution of the NO donor was added to a constantly stirring bulk solution (30 mL) for a final concentration of 0.1 mg mL-1. Of note, the larger volume of media was necessary to accommodate the working, reference, and counter electrodes in the flask.